PROJECT SUMMARY Chromosomal rearrangements are a great source of inter- and intra-specific genetic variation and are major contributors to human disease. Although position of rearrangement breakpoints can now be mapped at high resolution, interpreting the evolutionary or clinical implications of these events remains challenging. Depending on where they occur, rearrangements can disrupt the organization of genomic functional compartments, known as topologically associating domains (TADs). Within TADs, nearby loci (i.e. promoters and enhancers) interact frequently with each other, while interactions with loci outside TADs are prevented by TAD boundaries. Disruption of TAD boundaries can result in ectopic genes regulation, aberrant phenotypes, and genetic disorders. The functional outcomes of chromosomal rearrangements, therefore, can only be fully understood when studied in the context of genome topology. To shed light on some of the evolutionary implications of genome reorganization, we recently studied the gibbon genome, which has experienced rapid and recent karyotype evolution with respect to human and the other apes. In the gibbon genome, we observed that TADs remained genetically and epigenetically intact (genomic false-shuffle), because evolutionary breakpoints overlapped almost exclusively with TAD boundaries. Comparison with human and other mammals shows that these TAD boundaries are evolutionary conserved, indicating that TAD boundary establishment predated, and may have even contributed to, occurrence of evolutionary breakage. Motivated by our preliminary findings in gibbon, we propose to use a broad comparative and functional approach to assay multiple species with naturally highly rearranged genomes across the Boreoeutheria tree, and characterize the genetic context, epigenetic state, and evolutionary conservation of their TAD boundaries. We will determine if the false shuffle is a recurring mechanism of genome evolution and we will identify chromatin states associated with evolutionary fragility, as these regions and states could be relevant to human disease (Aim 1). Additionally, we will determine the level of conservation for TAD boundaries across different clades. Overall, the combination of these annotations will be a valuable resource to aid the interpretation of clinically and/or evolutionarily relevant rearrangements. We will then use an evolutionary-motivated approach to delete a subset of highly conserved and clade-specific TAD boundaries using CRISPR/Cas9 in cell lines and mouse, to assess the functional consequences of their deletion on DNA interaction, chromatin state, and gene expression (Aim 2). Finally, by analyzing differential gene expression and chromatin conformation between closely related species with structurally different genomes, we will evaluate the extent to which chromosomal rearrangements can alter short- and long-range functional interaction and contribute to differential gene expression (Aim 3). Overall, this study will elucidate the epigenetics changes associated with evolutionary genome reorganization and will help elucidating mechanisms by which genome rearrangement can lead to pathology.